Melatonin Improves the Resistance of Oxidative Stress-Induced Cellular Senescence in Osteoporotic Bone Marrow Mesenchymal Stem Cells

Accumulation of senescent bone marrow-derived mesenchymal stem cells (BMMSCs) has led to an age-related bone loss. However, the role of stem cell senescence in estrogen deficiency-induced osteoporosis remains elusive. Though melatonin plays a vital role in bone metabolism regulation, the underlying mechanisms of melatonin-mediated antiosteoporosis are partially elucidated. Therefore, this study purposed to explore (1) whether estrogen deficiency causes cellular senescence of BMMSCs, and if so, (2) the potential of melatonin in preventing bone loss via senescence signaling inhibition. BMMSCs derived from ovariectomized (OVX) rats (OVX BMMSCs) showed an impaired osteogenic capacity, albeit having comparable levels of senescence biomarkers than the sham cells. When exposed to low levels of hydrogen peroxide (H2O2), OVX BMMSCs rapidly exhibited senescence-associated phenotypes such as the increased activity of senescence-associated β-galactosidase (SA-β-gal) and upregulation of cell cycle inhibitors. Notably, the in vitro treatment with melatonin hindered H2O2-induced senescence in OVX BMMSCs and restored their osteogenic capacity. Treatment with either SIRT1 inhibitor (sirtinol) or melatonin receptor antagonists (luzindole and 4-P-PDOT) eliminated melatonin protective effects, thus indicating its potential in preventing stem cell senescence via SIRT1 activation through the melatonin membrane receptors. Following in vivo intravenous administration with melatonin, it successfully protected the bone microstructure and preserved the antisenescence property of BMMSCs in OVX rats. Collectively, our findings demonstrated that melatonin protected against estrogen deficiency-related bone loss by improving the resistance of BMMSCs to cellular senescence. Therefore, melatonin-mediated antisenescence effect on stem cells provides vital information to facilitate the development of a novel and effective strategy for treating postmenopausal OP.


Introduction
Estrogen deficiency is a major cause of postmenopausal osteoporosis (OP) that leads to an imbalance in osteoblastmediated bone formation and osteoclast-mediated bone resorption [1]. Bone marrow-derived mesenchymal stem cells (BMMSCs), the progenitor cells of osteoblasts, play a crucial role in mediating bone homeostasis. Based on emerging evidence, BMMSCs derived from OP patients exhibit degenerative properties such as a decreased proliferative capacity, impaired ability to migrate, and preference for adipogenic differentiation [2]. The senescent stem cells accumulated in the bone marrow with age are considered to be responsible for the age-related bone loss [3]. Cellular senescence is characterized by an irreversible proliferation arrest with distinctive phenotypic alterations such as enlarged cell sizes, resistance to apoptosis, permanent cell cycle arrest, and increased senescence-associated βgalactosidase (SA-β-gal) [4]. Besides, various types of stress can induce cellular senescence, such as telomere shortening, DNA damage, excessive reactive oxygen species (ROS), and oncogenic mutations [5]. The cell cycle-regulating factors involved in the senescence process include P53, P21 Cip1/Waf1 (P21), and P16 Ink4α (P16). Specifically, the accumulation of P16 triggers the onset of cellular senescence [6], while the elimination of P16-positive senescent cells in aged mice potentially promotes bone formation [7]. However, the role of stem cell senescence in estrogen deficiency-induced OP remains controversial. A study by Wu et al. reported that the BMMSCs from ovariectomized (OVX) rats had an increase in senescence biomarkers such as P53 and P16 [8].
Elsewhere, an emerging study showed contrary findings that elimination of senescent cells failed to rescue bone loss in OVX mice, thus indicating an independent role of cellular senescence in estrogen deficiency-induced OP [9].
Melatonin, mainly produced by the pineal gland, has been shown to play an important role in the regulation of bone metabolism [10]. Interestingly, during MSC osteogenesis, melatonin enhanced the expression of alkaline phosphatase (ALP) and matrix mineralization, even in the presence of proinflammatory cytokines [11]. Briefly, the intracellular signaling is transmitted via two high-affinity G protein-coupled receptors, MT1 and MT2 that are widely distributed in multiple tissues and organs [12]. MT1 receptors mediate the inhibition effect of melatonin on human breast cancer cells [13], whereas MT2 receptors are associated with osteoblast differentiation via the extracellular signal-regulated kinase (ERK)1/2 signaling cascade [14]. According to reports, oral administration of melatonin was able to effectively cure bone loss in OVX mice [15] and improve bone mineral density (BMD) at the femoral neck in postmenopausal women without major side effects [16].
Increased ROS with a simultaneous decrease in antioxidant enzymes were reported in OVX rats and postmenopausal women [17,18]. Oxidative stress induced by ROS generation and detoxication imbalance is a crucial factor in cellular senescence [19]. Overaccumulation of hydrogen peroxide (H 2 O 2 ) induces cellular senescence, therefore resulting to the decline in osteogenic differentiation of human MSCs [20]. Notably, melatonin protects MSCs against H 2 O 2induced senescence by upregulating the silent information of regulator type 1 (SIRT1), an important gene controlling cellular longevity [21]. In this study, we hypothesized that melatonin potentially ameliorated estrogen deficiencyinduced bone loss by preventing senescence of BMMSCs derived from OVX rats (OVX BMMSCs). Therefore, we exposed BMMSCs to sublethal concentrations of H 2 O 2 to induce senescence before evaluating the protective effects of melatonin. Subsequently, we injected OVX rats with melatonin via the tail vein and analyzed the antisenescence properties of BMMSCs.

Material and Methods
2.1. Animals. Eight-week-old female Sprague-Dawley (SD) rats (180 ± 13 g) were purchased from the Animal Center of Soochow University. The ovariectomy was performed on the rats following the standard method as previously described [22]. Bilateral OVX was carried out by the abdominal approach before excising ovaries from both sides of rats anesthetized with pentobarbital (30 mg/kg body weight, intraperitoneally; Yuanye, Shanghai, China). Similar procedures were followed in the sham group except ovaries were left intact. After the surgeries, the rats were sutured and injected with penicillin for three days (80,000 Units/rat, intramuscularly; Yuanye). Rats were housed under specific pathogen-free conditions with free access to water and fed on a standard laboratory rodent diet ad libitum. Animal experiments were performed following their approval by the Ethics Committee of Soochow University.

Isolation and Culture of BMMSCs.
Bone marrow cells were flushed out from the tibiae and femurs of OVX-or sham-operated rats, using alpha minimum essential medium (α-MEM, Thermo Fisher Scientific, Waltham, MA). Red blood cells were then removed using red blood cell lysis buffer (Beyotime Institute of Biotechnology, Haimen, China). After washing, bone marrow cells were incubated in a 75 cm 2 culture flask (Costar, Tewksbury, MA) containing α-MEM supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 μg/mL streptomycin (all from Thermo Fisher Scientific) and the cultures maintained at 37°C with 5% CO 2 for 3 days. The medium was changed, to remove nonadherent cells, and the adherent cells cultured in fresh medium. After attaining 80% confluence, 0.25% trypsin (Thermo Fisher Scientific) was added and the primary rat BMMSCs were replated. Subsequent experiments used cells that were from passage one.

2.3.
Treatments with H 2 O 2 , Melatonin, Compound C, Luzindole, 4-P-PDOT, and Sirtinol. To induce premature senescence, BMMSCs at approximately 50% confluence were exposed to H 2 O 2 for 2 h (Sigma-Aldrich, St. Louis, MO) and then cultured for an additional 3 days. Melatonin was dissolved in ethanol having a 250 mM stock concentration before diluted with α-MEM. Melatonin at 1 or 100 μM were incorporated into the culture medium after H 2 O 2 treatment. Cells in the control (CTRL) group were treated with an equal volume of the vehicle (0.4 μL ethanol/mL medium). BMMSCs were preincubated with 10 μM of compound C (CC, Sigma-Aldrich) for 2 h to inhibit AMPK phosphorylation. Melatonin receptors were blocked by treating BMMSCs with 10 μM of luzindole (an MT1/MT2 receptor antagonist) or 10 μM of 4-phenyl-2-propionamidotetralin (4-P-PDOT; a selective MT2 receptor antagonist). Moreover, SIRT1 was inhibited through treating BMMSCs with 40 μM of sirtinol (Sigma-Aldrich).
GACCCTCTCTCTGCTCACTCT GACCTTACTGCCCTCCTGCTTG 3 Oxidative Medicine and Cellular Longevity horseradish peroxidase-conjugated secondary anti-mouse antibody (1 : 5,000, ab6789, Abcam) or anti-rabbit antibody (1 : 10,000, ab6721, Abcam) for 1 h at room temperature, followed by visualization of bands using SuperSignal West Pico Substrate (Thermo Fisher Scientific) and X-OMAT BT Film (Beyotime). Gray values of the bands in scanned images were measured using ImageJ software (National Institutes of Health, Bethesda, MD), then normalized to that of α-tubulin before comparison.
2.11. Administration of Melatonin and Sirtinol In Vivo. OVX rats were injected with melatonin at 1 mg/kg (OVX+MT(L)) or 10 mg/kg (OVX+MT(H)) [23] via the tail vein for 3 months (twice per week). As controls, the sham and OVX groups were received saline containing the same amount of ethanol. The time for melatonin injection was between 10:00 and 10:30 AM daily with an infusion of about 5 seconds per rat to avoid possible physiological interference. Ovariectomized or shamop rats were subjected to melatonin in combination with sirtinol at 1 mg/kg to inhibit SIRT1 [24].
At each predefined time point, 1 mL of blood from the left ventricle of melatonin-treated rats was collected and centrifuged. The supernatant was immediately frozen at -80°C for further analysis. Subsequently, serum concentrations of melatonin were determined using a commercial ELISA kit (E-EL-R0031c, Elabscience Biotechnology, Wuhan, China) according to the manufacturer's instructions [25].
2.13. Histological Analysis. Femur specimens were fixed with 4% paraformaldehyde and decalcified in 10% ethylene diaminetetraacetic acid (EDTA) for six weeks. Bone samples were subsequently dehydrated in graded EtOH solutions, embedded in paraffin, and sectioned into 5 μm thick sections using a microtome. The paraffin sections were stained with hematoxylin and eosin (H&E) as previously described [26], and images captured using a bright field microscope (Zeiss Axiovert 200, Oberkochen, Germany).
2.14. Statistical Analysis. Data were presented as means ± standard errors of means (S.E.M.). One-way analysis of variance (ANOVA) was performed for multiple group comparison following the Tukey's post hoc test. Besides, independent two-tailed Student's t-test was used for two-group comparisons. Statistical analyses were performed using SPSS 13.0 statistical software (SPSS Inc., Chicago, IL) where * p < 0:05 and * * p < 0:01 were considered significant. Fig. 1A) showed that H 2 O 2 induced senescence in a dose-dependent manner. Notably, on H 2 O 2 exposure with the same concentrations (e.g., 68:5 ± 6:9% vs. 20:9 ± 2:8% at 100 μM), the percentage of β-gal-positive cells in the OVX group were markedly higher than the sham group. Since the sublethal dosage of H 2 O 2 at 100 μM had no serious apoptosis ( Supplementary Fig. 1B&C), it was selected for subsequent experiments. Cell cycle distribution indicated a G0/G1 cell cycle arrest in OVX BMMSCs due to the high proportion in the G0/G1 phase but low percentages in S phase ( Supplementary Fig 1D&E). Real-time PCR showed that exposure to H 2 O 2 significantly upregulated the transcript levels of P16, P21, and P53 by 1.0-fold, 2.3-fold, and 1.5-fold in OVX BMMSCs, respectively, while the mRNA level of Sirt1 was decreased by 73.1% (Figure 1(c)). Western blot assays confirmed that the protein expression of P16, P21, and P53 in the OVX+H 2 O 2 group was significantly increased, while SIRT1 expression was downregulated (Figures 1(d) and 1(e)). After a 14-day osteogenic induction, senescent sham and OVX BMMSCs showed decreased levels of both matrix mineralization (Figures 1(f) and 1(g)) and osteoblast-specific marker genes (Figure 1(h)). We also investigated the effect of melatonin on BMMSCs from sham-op rats. Exposure to 100 μM of H 2 O 2 resulted in 17:7% ± 2:4% β-gal-positive cells, whereas treatment with melatonin of 1 and 100 μM reduced the ratio of senescent cells to 11:1% ± 1:9% and 7:1% ± 1:2%, respectively (Supplementary Fig. 3A&B). The cell viability of H 2 O 2 -treated cells was increased by 100 μM of melatonin ( Supplementary Fig.  3C), and the gene expression levels of senescence markers such as P16, P21, and P53 were significantly downregulated ( Supplementary Fig. 3D). Meanwhile, treatment with melatonin improved the osteogenic differentiation of H 2 O 2 -treated BMMSCs, as evidenced by the enhanced matrix mineralization ( Supplementary Fig. 4A&B) and gene expression levels of osteoblast-specific markers ( Supplementary Fig. 4C).    (Figure 3(a)). Western blot results showed that melatonin treatment significantly increased the phosphorylation of AMPK and SIRT1 protein expressions (Figures 3(b) and 3(c)). The protein levels of P16, P21, and P53 in H 2 O 2 -treated OVX BMMSCs were significantly downregulated by melato-nin treatments, suggesting that melatonin prevents stem cell senescence through upregulation of SIRT1. To explore the AMPK pathway in melatonin-mediated antisenescence effect, OVX BMMSCs were treated with CC to inhibit p-AMPK before melatonin treatment. Inhibition of AMPK phosphorylation significantly decreased the mRNA level of Sirt1 by 27.2% (Figure 3(d)) and the protein level by 35.2% (Figures 3(e) and 3(f)).

Protection of OVX BMMSCs against Cellular
To investigate the involvement of melatonin receptors, two inhibitors luzindole and 4-P-PDOT were used before melatonin treatment. Consequently, the addition of luzindole downregulated the gene expression of Sirt1 39.8%,  7 Oxidative Medicine and Cellular Longevity whereas P21 was upregulated by 129.7% and 40.6% following the addition of both luzindole and 4-P-PDOT, respectively (Figure 4(a)). Western blot assays confirmed that either luzindole or 4-P-PDOT impeded the protective effect of melatonin, but the protein levels of MT1 and MT2 were not affected by melatonin receptor inhibitors (Figures 4(b)-4(d)). These results suggested that melatonin activated the AMPK-SIRT1 signaling pathway via melatonin receptors.

In Vivo Administration of Melatonin Ameliorated
Estrogen Deficiency-Induced Bone Loss by Preserving the Antisenescence Functions of BMMSCs. After intravenous injection with melatonin (1 and 10 mg/kg), serum concentrations of melatonin in OVX rats versus time were shown in Supplementary Fig. 5A. Melatonin administration successfully prevented bone deterioration in OVX rats ( Figure 5(a)). Three-dimensional reconstruction indicated that BMD and BV/TV in the OVX+MT (H) group was increased by  4) in qRT-PCR experiments. Statistically significant differences are indicated by * p < 0:05 or * * p < 0:01 between the indicated groups. Statistically significant differences are indicated by * p < 0:05 or * * p < 0:01 between the indicated groups; # p < 0:05 or ## p < 0:01 versus the CTRL group. 8 Oxidative Medicine and Cellular Longevity  Fig. 5B-E). The protective effect of melatonin on the bone micro-structure was determined by histological experiments (Figure 5(d)).
To further investigate the role of SIRT1, OVX or sham rats were injected with sirtinol and melatonin. The μCT results showed that the trabecular bone microstructure of melatonin-treated OVX rats was deteriorated following the sirtinol injection (Figure 8(a)) with 41.3% decrease in BMD (Figure 8(b)). However, sirtinol treatment consistently reduced BV/TV, BS/TV, and Tb.N, while increasing Tb.Sp and BS/BV in melatonin-treated OVX rats ( Supplementary  Fig. 8A-E). The results of H&E staining proved that sirtinol treatment terminated the protective effect of melatonin on bone microstructure (Figure 8(c)). Furthermore, the transcript level of Sirt1 was reduced by 54.8% in the MT+sirtinol group, while P16, P21, and P53 were significantly upregulated by 42.9%, 59.5%, and 24.4%, respectively (Supplementary Fig. 8F). The protein levels of SIRT1, P16, P21, and P53 were consistent with their gene expression (Figures 8(d) and 8(e)). The calcium deposition was decreased by 66.1% (Figures 8(f) and 8(g)), and the expression of osteoblast-    Oxidative Medicine and Cellular Longevity specific markers was also significantly downregulated following the sirtinol treatment ( Supplementary Fig. 8G). In addition, we examine the effect of sirtinol treatment on sham-op rats. As shown in Supplementary Fig. 9A&B, sirtinol significantly decreased BMD of sham-op rats with or without melatonin treatment. The evaluation of BMMSCs derived from sirtinol-treated sham rats demonstrated that sirtinol downregulated the expression of SIRT1, while increasing the expression of P16 and P21 ( Supplementary   Fig. 9C-E). Meanwhile, the cells showed attenuated osteogenic differentiation, as evidenced by the weak matrix mineralization and low expression of osteoblast-specific markers ( Supplementary Fig. 10).

Discussion
Previous studies have established that the biological properties of BMMSCs are altered in OP patients [22]. Consistent  Values are presented as the mean ± S:E:M of ten samples in each group (n = 10) in micro-CT and 3D reconstruction assays. Statistically significant differences are indicated by * p < 0:05 or * * p < 0:01 between the indicated groups; # p < 0:05 or ## p < 0:01 versus the sham group. 12 Oxidative Medicine and Cellular Longevity

13
Oxidative Medicine and Cellular Longevity with other studies, a significant decrease in the osteogenic capacity of OVX rat-derived BMMSCs was evident [27]. Based on studies, none showed estrogen deficiency directly induced stem cell senescence. On the contrary, a previous study has indicated that some senescence characteristics were detectable in OVX BMMSCs [8]. Briefly, exposure to a low level of oxidative stress rapidly induced senescence in OVX BMMSCs. Thus, to our knowledge, this is the first report to prove that estrogen deficiency results in a weak resistance of stem cells to oxidative stress-induced cellular senescence. Functionally, stress-induced senescence aggravates the impairment in BMMSC osteogenic capacity, leading to suppressed matrix synthesis and poor bone formation in OP patients.
From previous study findings, elimination of senescent cells in old mice not only improved bone formation but also suppressed bone resorption [7]. During the long-term expansion, melatonin supplementation preserved the functional   15 Oxidative Medicine and Cellular Longevity properties of stem cells and enhanced their therapeutic functions by inhibiting senescence phenotypes [28]. In response to some pathological stimuli such as iron overload [29] and uremic toxin exposure [30], melatonin effectively protects MSC differentiation potential by preventing premature senescence. In this study, in vitro treatments with melatonin successfully improved resistance of OVX BMMSCs to senescence. Furthermore, intravenous administration of melatonin significantly preserved their antisenescence properties, suggesting that melatonin-based therapy could be a promising strategy for treating postmenopausal OP patients.
Increased expression of P21 was reported in latepassaged MSCs whereas knockdown of P21 by shRNAs rescued their capacity for bone repair [31]. In this study, P21 was inhibited by melatonin in senescent BMMSCs, possibly through deacetylation of P53. The study of Han et al. showed that melatonin improved the functional survival of adipose-derived MSCs in infarcted hearts by decreasing Ac-P53 expression [32]. We observed that the P16 expression in senescent BMMSCs, another main regulator of cellular senescence [33], was also suppressed by melatonin. The nicotinamide phosphoribosyltransferase-(NAMPT-) SIRT1   (d, e) BMMSCs were isolated from melatonin-and sirtinol-treated OVX rats, exposed to H 2 O 2 (100 μM) for 2 h, and cultured for an additional 72 h. The protein levels of SIRT1, P16, P21, and P53 were determined using Western blot assays. (f) BMMSCs derived from melatonin-or sirtinol-treated OVX rats were induced toward osteogenic differentiation. Matrix mineralization was assessed by Alizarin Red S (ARS) staining. Scale bar =200 μm. (G) The stained mineral layers were quantified. The values shown were normalized to those of the sham group. Values are presented as the mean ± S:E:M of ten samples in each group (n = 10) in micro-CT and 3D reconstruction assays, four independent experiments (n = 4) in ARS assays, and three independent experiments (n = 3) in Western blot assays. Statistically significant differences are indicated by * p < 0:05 or * * p < 0:01 between the indicated groups; # p < 0:05 or ## p < 0:01 versus the sham group. 18 Oxidative Medicine and Cellular Longevity axis may be involved, both of which can be upregulated by melatonin [34] and overexpression of NAMPT was able to ameliorate senescence-associated phenotypic features in latepassaged MSCs [35]. However, further studies are needed to understand the different roles of melatonin in regulating P21 and P16 during stem cell senescence. SIRT1, an important target for energy metabolism, has been found to be involved in melatonin-mediated antisenescence effects. Activation of SIRT1 potentially improves the self-renewal capacity of BMMSCs by protecting sexdetermining region Y-(SRY-) box 2 (SOX2) from ubiquitination [36]. In vivo experiments confirmed that overexpression of SIRT1 in MSCs protected against bone loss in mice by enhancing the transcriptional activity of class O subfamily of forkhead box 3A (FOXO3A) [37]. Age-related senescence phenotypes in MSCs can also be attenuated by SIRT1 overexpression to improve telomerase activity and prevent DNA damage [38]. Therefore, our findings confirmed that melatonin prevents cellular senescence in OVX BMMSCs through SIRT1, because inhibition of SIRT1 by sirtinol terminated the protective effects of melatonin on H 2 O 2 -induced senescence. These results were consistent with a previous study that melatonin alleviated doxorubicin-induced acute cardiac dysfunction in mice through activation of AMPK, while blockade of AMPK compromised the cardio-protective action of melatonin [39].
To investigate the roles of MT1 and MT2 receptors in melatonin-mediated antisenescence, we treated OVX BMMSCs with either luzindole or 4-P-PDOT. Interestingly, both inhibitors counteracted the protective effects of melatonin, though 4-P-PDOT showed more potent effect than luzindole. According to previous studies, melatonin promoted bone formation through MT2 receptors and knockout of MT2 in mice resulting in a significant low bone mass rather than MT1 receptors [15]. However, the consistency in this study was possibly caused by estrogen deficiency that might affect the biological functions of melatonin through different receptors. Melatonin was shown to inhibit cell proliferation of human breast cancer cells by binding to MT1 receptors and suppressing estrogen-induced estrogen receptor alpha (ERα) transcriptional activity [40]. Estrogenstimulated mammary gland development was repressed by elevated MT1 receptor expression, suggesting that melatonin could modulate the estrogen response pathway through MT1 receptors [41]. Hence, the specific roles of both MT1 and MT2 receptors in modulating the biological functions of melatonin in estrogen-deficient patients are still unclear and recommended on their further explorations.
In addition to estrogen deficiency-induced OP, melatonin with antisenescence effects may benefit senile OP subjects. Senescent cells accumulated in the bone microenvironment drive bone loss when ageing [42]. Dietary melatonin supplementation can effectively improve the microstructure and biomechanical properties of bones in aged rats [43]. A possible mechanism is that melatonin protects the structural and functional integrity of vascular endothelium against ageing-  Figure 9: A schematic diagram illustrating the underlying mechanism of melatonin-mediated anti-senescence effect in OVX rats. Estrogen deficiency results in a bone loss and bone microstructure deterioration. BMMSCs derived from OVX rats (OVX BMMSCs) show that estrogen deficiency did not directly cause stem cell senescence, but exposure to low levels of oxidative stress rapidly induced premature senescence in OVX BMMSCs. Melatonin prevents oxidative stress-induced senescence in OVX BMMSCs and subsequently restores their impaired osteogenic capacity via activation of the AMPK-SIRT1 signaling pathway through melatonin receptors. Intravenous administration of melatonin ameliorates bone loss in OVX rats and preserves the anti-senescence property of BMMSCs.
19 Oxidative Medicine and Cellular Longevity induced damage [44]. However, another potential mechanism may involve its antioxidant properties. In elderly primary essential hypertensive patients, administration of 5 mg/day of melatonin improved their antioxidant defense functions, as demonstrated by a significant increase in superoxide dismutase (SOD) 1 and catalase activities as well as a reduction in the serum malondialdehyde level [45]. In OVX rats, melatonin attenuated ROS levels by upregulating mitochondrial antioxidant enzymes (e.g., SOD2 and glutathione peroxidase 1) [26]. Thus, the potential of melatonin to protect aged BMMSCs from senescence by improving their antioxidant functions necessitates further studies.

Conclusions
We demonstrate that estrogen deficiency did not directly cause stem cell senescence, but exposure to low levels of oxidative stress rapidly induced premature senescence in OVX BMMSCs. As illustrated in Figure 9, melatonin prevented oxidative stress-induced senescence in OVX BMMSCs and subsequently restored their impaired osteogenic capacity via activation of the AMPK-SIRT1 signaling pathway through melatonin receptors. Intravenous administration of melatonin-ameliorated bone loss in OVX rats and preserved the antisenescence property of BMMSCs. Herein, melatonin treatment represents a novel strategy for managing postmenopausal OP patients by enhancing the resistance of BMMSCs to cellular senescence.

Data Availability
The original contributions presented in the study are included in the article/supplementary material; further inquiries can be directed to the corresponding author.

Disclosure
This work was presented in Society for Redox Biology and Medicine (SfRBM) 27th Annual Conference in 2020 [46].